Dissecting the pathways controlling tunable responses to TCR signaling Summary Stimulation of the T-cell receptor (TCR) leads to activation, a process that includes changes in T cell metabolism, survival, proliferation, cytokine responsiveness, migration behavior, and effector functions. Life versus death, as well as lineage decisions of T cells are determined in large part by the strength of TCR signaling. Our previous studies have demonstrated that the transcription factor IRF4 is upregulated by TCR stimulation in CD8 T cells, and that the maximum level of IRF4 achieved is dependent on the strength of TCR signaling via the Tec kinase ITK. In turn, IRF4 promotes T cell differentiation into massively proliferating antiviral effector cells in a dose-dependent manner. Yet, mechanistic insight into how TCR stimulation produces a dynamic range of responses defined by distinct gene expression patterns is currently lacking. Our preliminary studies indicate that the Tec kinase ITK is a focal point for the tunable component of TCR signaling. Previous studies showed that ITK is not required for all TCR signaling; instead, in its absence, TCR signaling is significantly reduced. From these studies, the clear function of ITK was difficult to discern, as some aspects of T cell activation appeared normal in the absence of ITK, whereas other T cell functions were greatly impaired. Our current data, using IRF4 upregulation as an example of TCR tuning, provide a framework to understand these apparent discrepancies. These studies have revealed that variations in antigen density and in TCR affinity can modulate gene expression patterns shortly after activation of naive CD8 T cells. Dissecting these pathways shows that the two second messengers generated by activation of phospholipase C-?, the major substrate of ITK, cooperate to regulate all-or-nothing (digital) versus graded (analog) responses to changes in TCR signal strength. The relative balance of these differing inputs determines which responses exhibit the broadest range of tunability to TCR signaling. Based on these data, we hypothesize that the magnitude of ITK activity is determined by the multiplicity of ITAM phosphorylation at the TCR, and that variations in ITK activity tune the calcium response that regulates transcription factor activation. Further, we propose that ITK- dependent tuning of TCR signal strength controls a program of gene expression that is established within hours after TCR stimulation, and thus impacts the differentiation pathways of activated cells. To test these hypotheses, we propose to determine how variations in TCR signal strength lead to graded versus digital expression of entire programs of gene expression at the global and single cell level, and how these patterns are established by the first wave of transcriptional activation following T cell stimulation. We will also assess whether the magnitude of ITAM phosphorylation regulates the dynamic range of ITK-dependent TCR signaling. Finally, we will examine whether the full dynamic range of ITK-dependent TCR signaling is required to generate a broad repertoire of pathogen- specific T cells that provide enhanced responses to variants of the primary infecting pathogen. These experiments will provide important insights into the downstream consequences of alterations in TCR signal strength, allowing a more informed approach to manipulating T cell activation pathways for applications in the fields of vaccinology and immunotherapy.